Transcriptomic Effects of Adenosine 2A Receptor Deletion in Healthy and Endotoxemic Murine Myocardium

Clinical and Translational Science Institute Centers

3-1-2017

Transcriptomic effects of adenosine 2A receptor deletion in healthy and endotoxemic murine myocardium

Kevin J. Ashton Bond University

Melissa E. Reichelt The University of Queensland, Australia

S. Jamal Mustafa West Virginia University

Bunyen Teng West Virginia University

Catherine Ledent Universite Libre de Bruxelles

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Digital Commons Citation Ashton, Kevin J.; Reichelt, Melissa E.; Mustafa, S. Jamal; Teng, Bunyen; Ledent, Catherine; Delbridge, Lea M. D.; Hofmann, Polly A.; Morrison, R. Ray; and Headrick, John P., "Transcriptomic effects of adenosine 2A receptor deletion in healthy and endotoxemic murine myocardium" (2017). Clinical and Translational Science Institute. 528. https://researchrepository.wvu.edu/ctsi/528

This Article is brought to you for free and open access by the Centers at The Research Repository @ WVU. It has been accepted for inclusion in Clinical and Translational Science Institute by an authorized administrator of The Research Repository @ WVU. For more information, please contact [email protected]. Authors Kevin J. Ashton, Melissa E. Reichelt, S. Jamal Mustafa, Bunyen Teng, Catherine Ledent, Lea M. D. Delbridge, Polly A. Hofmann, R. Ray Morrison, and John P. Headrick

This article is available at The Research Repository @ WVU: https://researchrepository.wvu.edu/ctsi/528 Purinergic Signalling (2017) 13:27–49 DOI 10.1007/s11302-016-9536-1

ORIGINAL ARTICLE

Transcriptomic effects of adenosine 2A receptor deletion in healthy and endotoxemic murine myocardium

Kevin J. Ashton1 & Melissa E. Reichelt2 & S. Jamal Mustafa3 & Bunyen Teng 3 & Catherine Ledent4 & Lea M. D. Delbridge5 & Polly A. Hofmann 6 & R. Ray Morrison7 & John P. Headrick8

Received: 26 April 2016 /Accepted: 9 September 2016 /Published online: 30 September 2016 # Springer Science+Business Media Dordrecht 2016

Abstract Influences of adenosine 2A receptor (A2AR) activity implicating modified G protein/cAMP/PKA and cGMP/NOS on the cardiac transcriptome and genesis of endotoxemic myo- signalling. Lipopolysaccharide (LPS; 20 mg/kg) challenge for carditis are unclear. We applied transcriptomic profiling (39 K 24 h modified >4100 transcripts in wild-type (WT) myocardium

Affymetrix arrays) to identify A2AR-sensitive molecules, re- (≥1.5-fold change, FDR < 1 %); the most induced are Lcn2 vealed by receptor knockout (KO), in healthy and endotoxemic (+590); Saa3 (+516); Serpina3n (+122); Cxcl9 (+101) and hearts. Baseline cardiac function was unaltered and only 37 Cxcl1 (+89) and the most repressed are Car3 (−38); Adipoq

A2AR-sensitive genes modified by A2ARKO(≥1.2-fold change, (−17); Atgrl1/Aplnr (−14); H19 (−11) and Itga8 (−8). <5 % FDR); the five most induced are Mtr, Ppbp, Chac1, Ctsk Canonical responses centred on inflammation, immunity, cell and Cnpy2 and the five most repressed are Hp, Yipf4, Acta1, death and remodelling, with pronounced amplification of toll- Cidec and Map3k2. Few canonical paths were impacted, with like receptor (TLR) and underlying JAK-STAT, NFκBand altered Gnb1, Prkar2b, Pde3b and Map3k2 (among others) MAPK pathways, and a ‘cardio-depressant’ profile encompassing suppressed ß-adrenergic, PKA and Ca2+ signalling, electromechanical and mitochondrial function (and major Kevin J. Ashton and Melissa E. Reichelt denotes equal first authorship shifts in transcripts impacting function/injury including Lcn2, Electronic supplementary material The online version of this article S100a8/S100a9, Icam1/Vcam and Nox2 induction, and Adipoq, (doi:10.1007/s11302-016-9536-1) contains supplementary material, which is available to authorized users. Igf1 and Aplnr repression). Endotoxemic responses were selectively modified by A2AR KO, supporting inflammatory suppres- κ * John P. Headrick sion via A2AR sensitive shifts in regulators of NF B and JAK- [email protected] STAT signalling (IκBζ,IκBα, STAT1, CDKN1a and RRAS2) without impacting the cardio-depressant gene profile. Data indi- 1 Faculty of Health Sciences and Medicine, Bond University, Gold cate A2ARs exert minor effects in un-stressed myocardium and Coast, QLD, Australia selectively suppress NFκB and JAK-STATsignalling and cardiac 2 School of Biomedical Sciences, University of Queensland, injury without influencing cardiac depression in endotoxemia. Brisbane, QLD, Australia 3 Department of Physiology and Pharmacology, West Virginia Keywords Adenosine . Adenosine 2A receptor . University, Morgantown, WV, USA Anti-inflammatory . Cytokines . Endotoxemic myocarditis . 4 Universite Libre de Bruxelles, Brussels, Belgium Gene expression . Inflammation . Microarray . Sepsis 5 Department of Physiology, University of Melbourne, Parkville, VIC, Australia 6 Department of Physiology, University of Tennessee Health Science Introduction Center, Memphis, TN, USA 7 Division of Critical Care Medicine, St. Jude Children’s Research Uncontrolled inflammation with sepsis and the systemic inflam- Hospital, Memphis, TN, USA matory response syndrome induce multiple organ dysfunction, 8 Heart Foundation Research Center, Griffith University, including cardiac abnormalities key to disease progression and Southport, QLD 4217, Australia mortality [1]. Unravelling the complex mechanisms governing 28 Purinergic Signalling (2017) 13:27–49 myocardial injury [1, 2] is not only fundamentally important but [39, 40]. In vivo observations suggest marked expansion of also reveals targets for manipulating outcomes. In this regard, this signalling in intact myocardium [41, 42], contrasting adenosine 2A receptors (A2ARs) may fulfil a broadly suppressive in vitro evidence that cardiomyocyte NFκBandIκBkinase role to limit inflammatory injury in multiple tissues [3–6]and signalling is only transiently LPS responsive [43]. enhance myocardial resistance to ischaemic/hypoxic insult, pre- Importantly, these paths are inhibited by A2ARs in other cells, senting a potentially useful therapeutic target [3, 7]. In heart, this with exogenous agonism decreasing [44, 45]andA2ARKO G protein-coupled receptor (GPCR) influences coronary tone increasing NFκB activity [6]. Nonetheless, these paths can and angiogenesis, cardiac contractility, fibroblast growth and fi- also promote myocardial stress-resistance and survival under brosis and may mediate protection via ischaemic pre- and post- conditions that include inflammatory cytokine challenge conditioning [8–10]. Inflammatory modulation contributes to [46–48]. The molecular underpinnings of endotoxemic myo- this latter cardioprotection [9–11], together with the regulation carditis in vivo warrant further investigation, as do the mech- of myocyte kinase signals to limit oxidative stress, mitochondrial anisms countering this dysfunction (including A2ARactivity). dysfunction and cell death [12–14]. However, impacts of the We thus undertook broad-scale transcriptomic profiling of

A2AR on integrated myocardial responses to uncontrolled in- hearts from WTand A2AR KO mice, at baseline and following flammation, and the mechanisms underlying such effects, remain endotoxin challenge: shifts in gene expression can shed light to be elucidated. on both functions of the A2AR, and pathogenesis and modu- While adenosine analogues and A2AR agonists can limit lation of endotoxemic myocarditis. There are no prior analy- endotoxemic or septic injury in lung [15, 16], liver [17], brain ses of transcriptome-wide effects of A2ARactivityinmyocar- [18] and heart [19–21], the roles of intrinsic A2ARactivityare dium, and relatively few of the in situ cardiac response to less clear. Receptor deletion fails to modify inflammatory endotoxemia or sepsis [41, 42, 49]. markers/injury in some studies [22], reportedly worsens endotoxemic injury in heart [23] and lung [15, 24] and im- proves survival in models of polymicrobial sepsis [25, 26]. Materials and methods These divergent outcomes may reflect different cell- and organ-specific effects of A2ARs, for example promoting Animals inflammasome formation and macrophage-dependent injury [27], impairing bacterial clearance [25], while activating car- All animal procedures conformed to the Guide for the Care diac survival signalling and suppressing inflammation [3, 7, and Use of Laboratory Animals published by the NIH (publi- 12–14]. Nonetheless, opposing effects of endogenous vs. ex- cation number 85-25, revised 1996). This project (and proto- ogenous (or amplified endogenous) adenosine are evident cols for care of animals) was approved by the Animal Care within cell types, for example inhibiting vs. promoting vascu- and Use Committees of St. Jude Children’s Research Hospital lar myocyte NOS activation with inflammation [28]. The and the University of Tennessee Health Sciences Center. The chronicity of receptor activation may also be critical to A2AR KO mice and WT littermates were obtained from a sub- tissue-specific responses; while acute or transient increases colony of the original lines generated and characterized by in adenosine levels/receptor activity are generally beneficial Ledent et al. [50]. Analysis of cardiac tissue confirms ablation

(improving tissue perfusion, ischemic/hypoxic tolerance and of A2AR transcript without compensatory shifts in other aden- reducing cytokines/inflammation), chronic elevations may be osine receptors (Fig. S1). Mice were bred and maintained on- detrimental, exaggerating fibrotic processes via A2BRs in lung site at St. Jude Children’s Research Hospital, with standard [29]orA2A and A2BRs in liver [30, 31], for example. Whether laboratory food and water available ad libitum. Animal geno- acute vs. chronic A2AR activity induces opposing myocardial type was confirmed by PCR analysis of tail snips. All animals outcomes is unknown, though prolonged A2B agonism limits were originated from the same breeding series and were rather than promotes fibrosis in injured myocardium [32], as matched for age and weight. Male and female mice were used does prolonged A1 agonism [33]. with equal representation in each experimental group. Both Additional to questions regarding protective vs. deleterious A2AR KO and WT littermate mice were randomly allocated effects of the A2AR, the evolution of myocardial inflammatory to receive either an intraperitoneal injection of 20 mg/kg LPS injury and ‘endotoxemic myocarditis’ itself is complex and isolated from E. coli (Sigma-Aldrich, St. Louis, MO) or an incompletely defined [1, 2]. Effects in non-cardiac tissues in- equal volume of sterile saline vehicle. Blood was sampled at volve TLR/CD14-dependent NFκB and MAPK signalling 12 and 24 h of LPS challenge to assess shifts in blood cell and interferon/cytokine engagement of the JAK-STAT path counts and circulating cytokines/biomarkers, as detailed by us [34–36]. These mechanisms likely participate in heart, with previously [23]. Mice were monitored for the development of evidence NFκBandIκB kinase promotes cardiac dysfunction symptoms of illness or distress (lethargy, piloerection, in sepsis/endotoxemia [37, 38], while JAK-STAT signalling hunched posture and/or respiratory distress) during the initial mediates dysfunction and cell death in myocardial ischemia 12 h post-injection and every 4 h thereafter; observation of Purinergic Signalling (2017) 13:27–49 29 any combination of symptoms prompted immediate euthana- (Qiagen, Hilden, Germany). Total RNA yield and integrity sia. All efforts were made to minimize animal suffering and were determined spectrophotometrically and via capillary distress. No analgesic or anaesthetic was administered other electrophoresis on a 2100 BioAnalyzer (Agilent, Palo Alto, than for euthanasia immediately on evidence of illness/ CA), respectively. distress. Microarray experiments were performed at the St. Jude We initially tested responses to 24 h LPS challenge in both Hartwell Center Core Facility according to manufacturers’ young (14 week) and old (46–52 weeks) WT and A2ARKO protocols. Briefly, first- and second-strand complementary mice. However, absent overt symptoms of illness/distress, DNA (cDNA) synthesis reactions were performed using the there was nonetheless significant mortality in aged LPS- SuperScript Choice System (Invitrogen, Carlsbad, CA) treated mice, as detailed in the “Results” section and in followed by in vitro transcription using biotin-labelled Supplementary material. Cause of death in older animals dNTPs (ENZO Diagnostics, Farmingdale, NY). was not determined. As a result of mortality, detailed molec- Complementary RNA (cRNA) samples were fragmented ular interrogation of cardiac gene expression (n =6–8 per and individually hybridized to GeneChip® Mouse Genome group), together with analyses of cardiac function in isolated 430 2.0 microarrays (Affymetrix, Santa Clara, CA). Each mi- hearts (n =8–9 per group) and haematological/biomarker as- croarray quantified expression of over 39,000 transcripts, in- sessment (n =6–9 per group) is necessarily constrained to the cluding full-length mRNA sequences and expressed sequence young group of mice. tags (ESTs). Following hybridization, microarrays were washed and stained with streptavidin–phycoerythrin Langendorff perfusion and tissue sampling (Molecular Probes, Eugene, OR) before scanning. Image files were converted into probe-set data (*.CEL files) via After 24 h of LPS challenge, mice were anaesthetised with Affymetrix MAS 5.0 software. Experimental data are accessi- sodium pentobarbital (50 mg/kg intraperitoneally), a thoracot- ble through GEO Series accession number GSE44363 at omy performed and hearts excised into ice-cold Krebs- http://www.ncbi.nlm.nih.gov/geo. Henseleit solution for Langendorff perfusion, as detailed previously [23], or sampling of ventricular tissue for RNA prep- Microarray data and statistical analyses aration. Briefly, for perfusions, the aorta was immediately cannulated and hearts perfused at 80 mmHg with modified Raw expression data were background corrected, normal-

Krebs-Henseleit solution containing (in mM): NaCl, 120; ized and log2 transformed using the Robust Multichip NaHCO3, 25; KCl, 4.7; CaCl2, 2.5; MgCl2, 1.2; KH2PO4, Average (RMA) method in BioConductor/R [51]. Data 1.2; D-glucose, 15 and EDTA, 0.5. Perfusion fluid was main- were filtered to include only transcripts detected on ≥3 tained at 37 °C and bubbled with a mix of 95 % O2/5 % arrays, with 25,646 transcripts passing this quality criteri-

CO2 at 37 °C to provide a pH of 7.4. Ventricular function on (i.e. consistently detected in cardiac tissue). Log2 ratio was monitored via a fluid-filled plastic film balloon in the left values were generated by subtracting median expression ventricle, connected to a P23 XL pressure transducer (Viggo- values for WT vehicle-treated animals from each sample Spectramed, Oxnard, CA, USA). Coronary flow was moni- per probe before importing into TIGR MeV 4.0 software tored via a flow-probe in the aortic perfusion line, connected for statistical analysis [52]. The Significant Analysis of to a T206 flowmeter (Transonic Systems Inc., Ithaca, NY, Microarrays (SAM) algorithm was used to correct for USA). Functional data were recorded at 1 KHz on MacLab multiple comparisons and non-parametrically select differ- data acquisition system (ADInstruments, Castle Hill, entially expressed genes using a median false discovery Australia). Statistical comparisons of cardiovascular rate (FDR) ≤1.0 % [53]. In addition, a two-factor analysis (Table 1) and blood parameters (Figs. 1 and 2) between groups of variance (ANOVA) was performed. To generate a data were made via analysis of variance (ANOVA), with a sets similar in size and false-positive rates, a P value Newman-Keuls post hoc test for specific comparisons. A <0.005 was employed. The lists of differentially P < 0.05 was considered indicative of significance in all tests. expressed genes generated via SAM or ANOVA were then compared to identify 8110 shared transcripts for further RNA preparation and microarray hybridisation investigation and validation (Fig. S2). This output was subjected to appropriate pair-wise SAM comparisons, in-

Ventricular tissue was isolated, frozen in liquid N2 and stored corporating a 1.5-fold change cut-off [54]. Significant at −80 °C until analysis. Tissue was subsequently homoge- transcripts were annotated using Ingenuity Pathway nized in TRIzol reagent (Invitrogen, Carlsbad, CA) and total Analysis (IPA) (v8.7; Ingenuity® Systems, Redwood RNA isolated according to manufacturer’sprotocol.TheRNA City, CA, USA), providing insight into biological/ was further treated with 50 U of DNase I (Promega, Madison, molecular themes over-represented in response to A2AR WI) for 15 min at 37 °C and purified on RNeasy spin columns deletion and/or LPS. In brief, for each pathway/process, 30 Purinergic Signalling (2017) 13:27–49

Table 1 Ex vivo cardiovascular function in perfused for hearts Functional parameter WT (n =8) A2ARKO WT + LPS A2AR (n =8) (n =9) KO + LPS(n =9) from WT and A2AR KO mice

Coronary flow (ml/min/g) 16.7 ± 0.7 15.6 ± 1.2 16.9 ± 1.7 15.9 ± 0.6 Heart rate (beats/min) 341 ± 7 341 ± 7 352 ± 9 353 ± 9 Systolic pressure (mmHg) 113 ± 6 118 ± 5 80 ± 7* 86 ± 3* +dP/dt (mmHg/s) 6039 ± 247 6039 ± 163 4310 ± 422* 4489 ± 220* −dP/dt (mmHg/s) −3664 ± 129 −3718 ± 132 2666 ± 197* −2846 ± 105* Coronary ‘Supply:Demand’ (ml/ 0.15 ± 0.01 0.13 ± 0.02 0.21 ± 0.02* 0.18 ± 0.02* min/g/mmHg)

All data are means ± S.E.M. Functional parameters were measured after 30 min of normothermic aerobic perfusion. Intrinsic heart rate is recorded immediately prior to pacing. *P < 0.05 vs. corresponding values in Vehicle-treated hearts. Coronary ‘Supply:Demand’ was calculated as the ratio of coronary flow rate (O2 supply) relative to ventricular systolic pressure (reflecting myocardial O2 demand). dP/dt, first derivative of pressure over time. *P < 0.05 vs. untreated. A2AR KO did not independently modify functional parameters the fraction of differentially expressed genes was com- in specific datasets). The probability of involvement of pared with the fraction of total genes in that path (shown the respective number of modified transcripts in a in tables as the ‘Ratio’, useful for determining which path/processisexpressedasaP value or range (values pathways/processes overlap the most with altered genes <0.05 considered significant).

Validation of expression changes and Adora expression via RT-qPCR

Two-step RT-qPCR, utilizing SYBR Green I, was employed to confirm differential gene expression for 11 transcripts (primer details provided in Table S1), as previously described [54]. Six additional genes (Actb, Gapdh, Hprt1, Pgk1, Ppia and Ubc) were assessed using GeNorm to determine utility as reference genes [55]. Following GeNorm assessment, Actb was found to be most stable (M = 0.03) and served as the endogenous reference control for all messenger RNAs (mRNAs) assessed via RT-qPCR. Briefly, 1 μg total RNA was used to synthesize cDNA using the Superscript III First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA) according to manufacturer’s instructions. RT-qPCR was performed in a CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA). Final reaction volumes (10 μL) included 5 μL iQ SYBR-Green Supermix (Bio- Rad, Hercules, CA), 100 nM of each primer and 4 μLofa 1:20 dilution of cDNA. Optimal qPCR cycling conditions entailed an initial denaturation at 95 °C for 3 min followed by 40 cycles of 95 °C for 15 s/62 °C for 60 s. After the final PCR cycle, reactions underwent melt curve analysis to detect non-specific amplicons. All reactions were performed in triplicate, with each plate containing an equal number of samples from each group, a calibrator control derived from a pool of all cDNA samples and a no- Fig. 1 Effects of A2AR KO and LPS (24 h) on markers of cardiac template control. PCR amplification efficiencies (90– damage (TnI) and systemic inflammation (CRP) and the acute phase response (haptoglobin). Data represent means ± S.E.M. *P <0.05vs. 110 %) for each primer pair were calculated using a 5- vehicle; †P < 0.05 vs. wild-type log serial dilution of calibrator sample. PCR data were Purinergic Signalling (2017) 13:27–49 31

Fig. 2 Effects of A2AR KO and LPS (24 h) on haematological parameters and cytokines. Data represent means ± S.E.M. *P < 0.05 vs. vehicle; †P <0.05 vs. wild-type analysed using CFX Manager v1.6 (Bio-Rad, Hercules, not modify cardiac or coronary function in healthy or CA). Baseline subtractions and threshold settings above endotoxemic hearts (Table 1). Cardiovascular, blood cell background were applied to all data. The calibrator sam- and cytokine responses to LPS in WT and A2ARKO ple was used to normalize inter-assay variations, with the mice have been reported by us previously [23]. Similar threshold coefficient of variance for intra- and inter-assay outcomes were apparent here for markers of inflamma- replicates <1 % and <5 %, respectively. Normalized ex- tion and injury (Fig. 1), haematological parameters and pression (ΔΔCq) was calculated, with mRNAs normal- cytokines (Fig. 2) and cardiac function (Table 1). Data ized to Actb levels and the calibrator control then log2- confirm significant inflammatory activation, cellular in- transformed. jury and cardiac dysfunction with LPS, with A2ARde- letion amplifying changes in IL-5 and markers of cardiac injury (TnI) and systemic inflammatory stress (haptoglobin, CRP), without altering myocardial dysfunction Results or circulating levels of IL-4, IL-10, IFNγ or TNFα.

Impact of A2AAR deletion on cardiovascular function and inflammatory markers Impact of A2AAR deletion on survival

Deletion of the A2AR was confirmed in cardiac tissue, Interestingly, we acquire preliminary evidence LPS- with no compensatory changes in transcription of the dependent mortality is age- and sex-dependent and selectively other three sub-types (Fig. S1). Receptor deletion did exaggerated by A2AR KO in older males (Fig. S3). Initially, 32 Purinergic Signalling (2017) 13:27–49 testing responses to LPS challenge in young (14 week) and functions (Table 4). The latter included cardiac arteriopathy middle-aged (46–52 week) mice—since age may exaggerate (Ank3, Pon1, Cux2, Gk5, Pde3b, Dgat1 and Nsmce1), infarc- impacts of endotoxemia [56]—we recorded 20 % mortality in tion (Pon1 and Acta1) and transcripts involved in failure and older WT mice with A2AR KO specifically reducing survival hypertrophy (Pde3b and Nfat2c). to <20 % in older males (Fig. S3). No mortality was recorded in young mice challenged with LPS (or any vehicle-treated Cardiac transcriptomic response to LPS groups). There is thus a trend to greater mortality with age, with a major survival effect of A2AR activity in older males. Analysis via the SAM algorithm (1.5-fold threshold, 1 % While molecular interrogations here are thus limited to young FDR) identified 4146 transcripts modified after 24 h of LPS mice (in which LPS was non-lethal), it is possible A2ARac- challenge (see Table S4 for full details). Figure 3 presents the tivity may be increasingly crucial to survival in older, stress- 25 most induced and repressed transcripts, with responses in intolerant males (whereas younger animals and females may A2AR KO hearts shown for comparison. Many of the most possess intrinsically greater resistance to injury/death [57]). highly modified are predictably involved in inflammation/ This is discussed further in the online Supplementary material. innate immunity, together with tissue development/remodelling. The most LPS-responsive cytokines/chemokines are

Cardiac transcriptomic response to A2ARKO shown in Fig. 4, with responses in KO hearts highlighted. Functional classifications, using a twofold threshold to nar- Few transcriptional differences were detected between WT row the focus to the most highly modified paths, identify 236 and A2AR KO hearts, with the amplitude of changes modest canonical pathways (Table S5), with the most significantly (up to threefold). Employing an initial 1.5-fold cut-off and 1 % modified presented in Table 5. Data confirm a profound in- FDR identified only 13 genes (Table 2). To enhance the power flammatory and immune response, with marked upregulation of subsequent pathway analysis, these criteria were relaxed to of TLR/MyD88 and interferon signalling (Fig. 5), the acute 1.2-fold and 5 %, encompassing 37 altered transcripts phase response (Fig. S4), IRF activation (Fig. S5) and PRR-

(Table 2). There was a little impact of A2ARdeletiononin- and RIG-1-like signalling (Figs. S6 and S7). These changes flammatory mediators, with modest induction of a CXC che- primarily reflect shifts in underpinning NFκB, JAK-STAT, mokine (Ppbp, +2.1), a regulator of cell migration/adhesion MAPK and PI3K/Akt signalling (Figs. S8–S12). and cytokinesis (Iqgap1, +1.6) and a hemopoietic cytokine Additionally, LPS upregulated cell death signalling (Tslp, +1.6), together with repression of Hp (−2.7) and an Ig (Figs. S13 and S14) and modified paths involved in cellular adhesion molecule regulating T-lymphocyte development differentiation, movement, growth and remodelling (e.g. (Mpzl2, −1.7) (Table 2). Figs. S15 and S17). The top LPS-sensitive biological func- Analysis via the IPA suite identified 112 canonical path- tions are summarized in Table S6, with biologic and cardio- ways sensitive to A2AR activity in healthy hearts (Table S2), vascular toxicological responses fully detailed in Table S7 and the most highly modified shown in Table 3. The top five S8 in the Supplementary material. included relaxin signalling, cardiac β-adrenergic signalling, Unsurprisingly, many highly responsive transcripts were cellular effects of sildenafil, PKA signalling and photo-trans- involved in inflammation (Figs. 3 and 4, Table 5): within the duction. The influence of A2AR KO on these and other paths most induced are interferon-related genes (Ifit1, Ifit2, Ifit3, can be attributed to repression of Prkar2b (−1.8), Gnb1 (−1.7) Igtp1, Igtp2, Igtp3, Iigp1, Iigp2, Ifi44 and Irf7)and and Pde3b (−1.7), collectively impacting G protein-coupled inflammatory/immune modulators (Lcn2, Saa3, Socs3, cAMP/PKA dependent signalling (encompassing relaxin, α- S100a8, S100a9, Mpa2l, Cxcl1, Cxcl9, Cxcl10, IL6, Ptx3, and β-adrenergic, NO, Ca2+ and hypertrophic signalling). Serpina3n, Csf3 and Mt2). Several are potentially injurious Additionally, Map3k2 (−2) and Nfat2c (+1.3) span many paths to the heart: the most highly induced transcript encodes modified by A2AR KO. Modulation of these five transcripts lipocalin-2, which regulates inflammation and matrix degra- contributes to 48 of the top 50 A2AR-sensitive canonical path- dation, is implicated in heart failure [58] and promotes cardiac ways. Overall, A2AR deletion exerts an inhibitory effect on G apoptosis [59]; CXCL1 is a potent neutrophil chemoattractant; protein-coupled, cAMP/PKA, and MEKK2 signalling down- CXCL9 stimulates cytokine production and T-cell stream of this and other GPCRs. proliferation/recruitment; CXCL10 mediates CXCR3+ cell

Multiple biological functions appear sensitive to A2ARKO migration and augments inflammation. Conversely, some (see Table S3), the most significant (Table 4) revolving around transcripts encode anti-inflammatory and potentially protec- cellular development, growth, movement and death, together tive molecules: SERPINA3N (α1-antichymotrypsin) inhibits with humoral immunity and haematological development and proteases involved in inflammation; the pentraxin PTX3 is intercellular signalling. Toxicological functions included high expressed in heart with inflammation and limits cell death; representation of liver processes (fibrosis, damage, steatosis, SOCS3 is a negative feedback regulator of IL-6 signalling, hepatitis and inflammation) together with six cardiac implicated in sex-dependent cardiac stress-resistance; Purinergic Signalling (2017) 13:27–49 33

Table 2 Genes modified by A2AR KO in healthy myocardium

Gene Gene name Affymetrix ID Fold-Change %FDR

UpRegulated Mtr 5-methyltetrahydrofolate-homocysteine methyltransferase 1439811_at 2.83 0.00 Ppbp pro-platelet basic protein (chemokine (C-X-C motif) ligand 7) 1418480_at 2.08 3.93 Chac1 ChaC, cation transport regulator homologue 1 (E. coli) 1451382_at 1.85 0.00 Ctsk cathepsin K 1450652_at 1.84 0.00 Cnpy2 canopy 2 homologue (zebrafish) 1416507_at 1.67 0.00 Iqgap1 IQ motif containing GTPase activating protein 1 1445724_at 1.64 3.85 Tslp thymic stromal lymphopoietin 1450004_at 1.63 3.93 Nav3 neuron navigator 3 1456144_at 1.61 0.00 Cux2 cut-like homeobox 2 1447500_at 1.60 0.00 Slc38a1 solute carrier family 38, member 1 1454764_s_at 1.47 0.00 Nsmce1 non-SMC element 1 homologue (S. cerevisiae) 1436121_a_at 1.42 0.00 Gk5 glycerol kinase 5 (putative) 1436210_at 1.38 3.93 Rpl22 ribosomal protein L22 1448398_s_at 1.37 0.00 C11orf75 chromosome 11 open reading frame 75 1419403_at 1.37 0.00 Slco5a1 solute carrier organic anion transporter family, member 5 A1 1440874_at 1.31 4.21 Nfatc2 nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 2 1426032_at 1.28 4.70 Downregulated Dgat1 diacylglycerol O-acyltransferase homologue 1 (mouse) 1418295_s_at −1.24 2.39 Ticam1 toll-like receptor adaptor molecule 1 1454676_s_at −1.24 3.05 Mrap melanocortin 2 receptor accessory protein 1451371_at −1.31 3.05 Ppa1 pyrophosphatase (inorganic) 1 1416939_at −1.32 4.31 Ell2 elongation factor, RNA polymerase II, 2 1450744_at −1.33 2.53 Fmod fibromodulin 1456084_x_at −1.47 4.31 Ptger3 prostaglandin E receptor 3 (subtype EP3) 1450344_a_at −1.49 2.68 Pkdcc protein kinase domain containing, cytoplasmic homologue (mouse) 1454838_s_at −1.50 4.61 Ank3 ankyrin 3, node of Ranvier (ankyrin G) 1457288_at −1.58 3.05 Sacs spastic ataxia of Charlevoix-Saguenay (sacsin) 1434958_at −1.61 2.39 Pon1 paraoxonase 1 1418190_at −1.68 0.00 Gnb1 guanine nucleotide binding protein (G protein), beta polypeptide 1 1425908_at −1.72 4.54 Mpzl2 myelin protein zero-like 2 1416236_a_at −1.74 0.00 Pde3b phosphodiesterase 3B, cGMP-inhibited 1433694_at −1.74 0.00 Prkar2b protein kinase, cAMP-dependent, regulatory, type II, beta 1438664_at −1.78 3.05 Slc16a9 solute carrier family 16, member 9 (monocarboxylic acid transporter 9) 1429726_at −1.84 0.00 Map3k2 mitogen-activated protein kinase kinase kinase 2 (MEKK2) 1438719_at −2.01 0.00 Cidec cell death-inducing DFFA-like effector c 1452260_at −2.08 0.00 Acta1 actin, alpha 1, skeletal muscle 1427735_a_at −2.14 2.68 Yipf4 Yip1 domain family, member 4 1426417_at −2.51 4.54 Hp haptoglobin 1448881_at −2.70 0.00

Data shown for transcripts for which myocardial expression levels were modified by A2AR KO by a factor of ≥1.2-fold (FDR <5 %) metallothionein-2, induced by STAT3-related signalling, me- Adrenergic signalling was broadly suppressed diates cardioprotection and can limit dysfunction in sepsis. (Fig. S18), including reduced transcripts for β1-adrenergic receptor, G protein, adenylate cyclase, PKA and related Cardio-depressant molecular profile Major pathways crit- signalling elements (Ppp1r14c, Gng11, Prkar2b, Pde7a, ical to contractile function were substantially impacted, Ppp1r3c, Ppp1r14a, Pkia and Adcy7), together with in- identifying a ‘cardio-depressed’ molecular profile in duction of Pde4b (AMP-dependent phosphodiesterase). hearts of endotoxemic mice (Figs. S18–S21). β- The PKA pathway itself was substantially suppressed 34 Purinergic Signalling (2017) 13:27–49

(Fig. S19), including reductions in PKA RIα (Prkar1a), A2AR KO (Table S9). Effects of LPS on 282 genes were PKA RIIα (Prkar2a), PKA RIIβ (Prkar2b), PKAα augmented ≥1.5-fold following A2AR KO, with a further 9 (Prkaca) and PKA-Cβ (Prkacb), while PKA RIβ responsive to LPS specifically in KO and not WT hearts (Prkar1b) was modestly induced (+1.5). Transcripts for (Table S9). The most highly augmented included 25 tran-

Gαs and Gαq also declined. scripts increased ≥twofold by KO. Many A2AR-sensitive tran- Cellular Ca2+ signalling paths were repressed (Fig. S20), scripts are associated with pro-inflammatory signalling (e.g. with reductions in key elements including: Camk1d, Ryr3, Igtp, Lcn2, Cxcl5, S100a8, Iigp2, Cxcl2, Il6, Ifi202b, Hp and

Casq1, Mef2c, Casq2, Calm1, Hdac9, Myh7b, Tpm2, Cxcl9), with data supporting inhibitory effects of the A2ARon Myh11, Rcan1, Myl7, Atp2b2, Prkar2b, Tpm3 and Asph. the responsiveness of canonical pathways that include the Mitochondrial dysfunction (Fig. S21) is also evidenced by acute phase response, glucocorticoid receptor, Erk/MAPK, repression of complex I/NADH dehydrogenase components HIF1α and JAK-STAT signalling (Table 6).

(including Ndufv2 and 3; Ndufs4, 5, 6, 7 and 8; Ndufa3, 4, 5, 6 Deletion of the A2AR reduced or entirely eliminated 134 and 8; Ndufab1, Ndufaf1, Ndufb2, 3, 4, 7, 9, 10 and 11), gene responses to LPS (Table S9). Responses entirely A2AR- together with Complex II components (including Sdhc, dependent included repression of Sacs, Cidec, Gnb1l, Sdhd, Uqcrb, Uqcrc1 and Uqcrfs1). Uncoupling proteins Mtap1b, Msi2, Cdh2, Lpgat1, Tbx3, Slc38a1, Sdc1, Reln, Ucp2 and Ucp3 were also induced, while Ucp1 was repressed. Ext1 and Pvrl3, and induction of Rit1, Ezh1, Eif4ebp1 and

Transcript for the major activator of mitochondrial biogenesis Abhd4 (Table S9). The transcripts most sensitive to A2AR ( Ppargc1a) was repressed. KO included Dnmt3a, Qk, Sema5a, Srpk2, Car3, Ucp1, Rsad2 and Dnajb14. Highly LPS-responsive changes

Adenosine-related transcripts There were limited impacts on counteracted by A2AR KO included: Ifit3, Rsad2, Cxcl13, the adenosinergic system itself, with two- to threefold upregu- Ms4a6d, Oasl1, Ms4a4c, Sema5a, Tyki and Hmox1.Intrinsic lation of adenosine deaminase, 1.5-fold repression of adenosine A2AR activity thus also promotes LPS-dependent shifts in kinase, 1.7-fold induction of S-adenosylhomocysteine hydro- select genes associated with immune/inflammatory processes, lase and twofold induction of the ecto-nucleotidase CD39. including orphan nuclear receptor and PPARα activation, These changes may modify patterns of cellular adenosine gen- PRR responses to infection, AMPK signalling and NFκBsig- eration vs. uptake and metabolism, potentially altering receptor nalling (Table 6). activation in endotoxemic tissue. A small 1.3-fold rise in In terms of biological functions, A2AR activity reduced Adora2b expression was also detected. LPS responsiveness of paths related to cellular movement, growth, signalling and death, immune cell trafficking and

Impact of A2AR KO on transcriptomic responses haematological and cardiovascular development (Tables 7 to endotoxemia and S10). Conversely, A2AR activity appears to augment responses in pathways related to metabolism and trans-

Interestingly, A2AR activity does not broadly suppress the port, together with organ, haematological, cardiovascular effects of endotoxemia on myocardial gene expression, with and lymphoid development (Table 7), with complete de-

90 % of transcriptomic responses unaltered by receptor KO. tails of pathway responses modified by A2ARKOinclud- For example, Fig. 3 presents response profiles for the 25 most ed in Table S10. Deletion of the A2AR also influenced induced or repressed transcripts in WT hearts, with most multiple toxicological functions; of 35 responses coun- shown to be insensitive to A2AR KO. Deletion selectively teredbyA2AR KO, 12 were cardiac-specific categories enhanced induction of Lcn2, Igtp, Cxcl5, S100a8, Iigp2, (Table S11), with reduced Adipoq and Hmox1 responses Cxcl2 and Iigp1 andrepressionofC1qtnf9, Colec11 and involved in ~half. Of 46 toxicological function responses

Inmt, while reducing induction of Ifit3 andrepressionof enhanced by A2AR KO, a third was cardiac categories. Adipoq, Gpr22,andScube2, Ifit3 (Fig. 3). These effects sup- Enhanced responses for Serpine1, Kckn3 and Ppargc1a port A2AR modulation of inflammatory and interferon-related are involved in a majority of these and are also implicated signalling responses. Nonetheless, the pattern of LPS- in cardiac dysfunction. dependent cytokine/chemokine change was largely insensitive Importantly, a relatively small set of A2AR responsive to A2AR KO, with specific enhancement of Cxcl5, Cxcl2, Il6 genes are implicated in a majority of these A2ARsensitive and Csf3 induction (Fig. 4). Figure 6 summarizes the select biological pathways and processes and can be considered effects of A2AR activity (revealed via KO) on TLR- and A2AR-dependent ‘nodes’ or points of regulatory conver- interferon-triggered NFκB and JAK-STAT signalling re- gence. Of the top 50 pathway responses augmented by sponses to LPS (generally repressing these pathways). A2ARKO,Pi3kr1 is implicated in 42; Rras2 in 40; To gain further insight into these specific effects of A2AR Stat1, Vegfc, Pgf and Pak6 each in 12; Atf4 in 11; KO, we identified those LPS-responsive transcripts whose Cdkn1a in 9 and Rhoj and IL6 each in 8 (Tables 6 and expression changes were increased or reduced ≥1.5-fold by S10). Of the leading 50 canonical responses inhibited by Purinergic Signalling (2017) 13:27–49 35

Table 3 Top 20 canonical pathways modified by A2AAR KO in healthy myocardium

Canonical pathways P value Ratio Genes

Relaxin signalling 3.55E-03 1.99E-02 Gnb1,Prkar2b,Pde3b Cardiac β-adrenergic signalling 3.55E-03 2.11E-02 Gnb1,Prkar2b,Pde3b Cellular effects of sildenafil (Viagra) 3.98E-03 1.99E-02 Acta1, Prkar2b, Pde3b Protein Kinase A signalling 6.03E-03 1.26E-02 Gnb1, Prkar2b, Pde3b, Nfatc2 Phototransduction pathway 6.03E-03 3.08E-02 Gnb1, Prkar2b Germ cell-sertoli cell junction signalling 6.31E-03 1.88E-02 Iqgap1, Acta1, Map3k2 Calcium signalling 8.71E-03 1.47E-02 Prkar2b, Nfatc2, Acta1 Glutamate receptor signalling 9.55E-03 2.86E-02 Gnb1, Slc38a1 Caveolar-mediated endocytosis signalling 1.41E-02 2.35E-02 Acta1, Map3K2 Nitric oxide signalling in the cardiovascular system 1.48E-02 2.02E-02 Prkar2b, Pde3b Leptin signalling in obesity 1.55E-02 2.38E-02 Prkar2b, Pde3b Cardiac hypertrophy signalling 1.78E-02 1.22E-02 Gnb1,Prkar2b,Map3k2 TR/RXR Activation 2.09E-02 2.02E-02 Hp, Pde3b SAPK/JNK signalling 2.14E-02 1.98E-02 Gnb1, Map3k2 Colorectal cancer metastasis signalling 2.14E-02 1.17E-02 Gnb1,Prkar2b,Ptger3 α-Adrenergic signalling 2.19E-02 1.89E-02 Gnb1, Prkar2b RANK signalling in osteoclasts 2.19E-02 2.04E-02 Nfatc2, Map3K2 G Beta Gamma signalling 2.24E-02 1.68E-02 Gnb1, Prkar2b IL-1 signalling 2.29E-02 1.89E-02 Gnb1, Prkar2b fMLP signalling in neutrophils 2.95E-02 1.57E-02 Gnb1, Nfatc2

Canonical signalling paths modified by A2AR KO are shown, ranked according to P values determined by a Fisher’s Exact Test. Also shown is a ratio value reflecting the number of molecules in a given path that meet cut-off criteria for differential expression, divided by the total number of molecules in the pathway

A2ARKO,Rac2 was implicated in 34, Nfkbie in 21, between both measures (with a slope > 1 suggesting Prkar2b in 10, Hmox1 and Eif2ak2 eachin6and greater dynamic range for RT-qPCR measurement). Eif4ebp1 and Cyp2b6 each in 5 (Tables 6 and S10).

These data point to an influence of intrinsic A2ARactivity on key signalling responses, including G protein, PKA, Discussion STAT1 and NFκ B-dependent processes (see

Supplementary material for additional discussion). While The present study characterizes impacts of A2AR dele- the absence of A2ARs impacted inflammatory and under- tion on the myocardial transcriptome, markers of inflam- lying signalling changes in endotoxemia, paths implicated mation and cardiac function and injury in healthy and in endotoxemic cardio-depression (ß-adrenergic and PKA endotoxemic mice. Data indicate A2ARactivityexerts signalling, Ca2+ handling, excitation-contraction coupling limited effects in un-stressed myocardium, with and mitochondrial function; Figs. S18–S21)werenot transcriptomic changes limited to G protein, among those sensitive to A2ARKO(Table6), consistent cAMP/PKA and cGMP/NO signalling downstream of with a lack of effect of A2AR KO on contractile dysfunc- this and other GPCRs (Tables 2 and 3). However, dur- tion itself (Table 1). ing endotoxemia, absence of A2ARs exaggerated myocardial injury (and age- and sex-dependent mortality), without substantially modifying patterns of cytokine re- PCR analysis of select transcripts lease, myocardial cytokine/chemokine transcription or contractile depression. The latter is consistent with in- Quantitative RT-PCR analysis supports microarray- sensitivity of the ‘cardio-depressant’ profile in determined changes in transcripts selected for differen- endotoxemic hearts to A2AR KO. Rather, data reveal tial responsiveness to LPS and A2ARKO(Fig.7). A2AR activity selectively influences transcription of reg- While expression ratios vary slightly between the two ulators of NFκB and JAK-STAT signalling during methods, a significant linear relationship was apparent endotoxemia, which may limit myocardial inflammation 36 Purinergic Signalling (2017) 13:27–49

and injury. Additional changes with A2AR KO suggest cardiomyocyte [43, 49] responses to endotoxin/sepsis. potential influences on insulin-resistance, hypertrophy/ Approximately 15 % of the 25,646 transcripts expressed remodelling and vascular control/angiogenesis in in murine hearts were modified in endotoxemia endotoxemia. (Table S4), encompassing a multiplicity of canonical paths and functions (Tables 5, S5–S7). Many are con-

A2AR activity and the transcriptome in un-stressed sistent with those highlighted by Wong et al. in more myocardium limited analysis of endotoxemic rats [41] and Rudiger et al. in a rat faecal peritonitis model [49]. The myo-

Modest impacts of A2AR KO in un-stressed hearts cardial response involves profound upregulation of (Table 2) are consistent with a largely retaliatory or immune/inflammatory paths (Figs. 3–5 and S4–S7), the stress-responsive role for myocardial A2ARs. Indeed, de- most highly modified including acute-phase response, letion failed to modify cardiac or vascular function, and PRR/TLR and interferon/IRF signalling (Table 5). circulating CRP, haptoglobin and cytokines in healthy an- Underlying NFκB, JAK-STAT and MAPK/PI3K paths imals. Functional annotation of transcripts supports A2AR- are up-regulated (Tables 5, S5 and S7;Figs.S8–S12), dependent shifts in relaxin, adrenergic, Ca2+, PKA, with NFκB/JAK-STAT mechanistically linking the top SAPK/JNK and hypertrophic pathways (Tables 3 and five canonical pathway responses. Integrated signalling S2), involved in cellular growth/movement/death, immune via NFκB and JAK-STAT paths thus appears central to and cell-to-cell signalling, and toxicological processes of myocardial endotoxemia, as in other tissues. In other fibrosis, cell damage and inflammation. This profile stems tissues, these paths are also targeted by A2ARs to sup- from a handful of changes spanning pathways (i.e.Gnb1, press inflammatory/immune responses [44, 45, 58], with Nfat2c, Acta1, Prkar2b, Map3k2 and Pde3b), supporting KO augmenting NFκB activation in macrophages, for effects of A2AR activity on G protein and cAMP/PKA example [6]. signalling downstream of the receptor [5, 8, 60].

Deletion of the A2AR has been shown to reduce cAMP Amplified cardiac TLR and interferon signalling Since and PKA activation in other cell types [61], consistent uncontrolled inflammation induces cellular injury/death, with impacts of KO here (Table 3). The altered MEKK2 pro-inflammatory TLR/CD14 signalling is the subject to path is also linked to G protein/Rac-dependent signalling negative control. However, this path was transcriptional- distal to this and other GPCRs. In terms of vasoregulatory ly amplified in endotoxemic myocardium (Fig. 5), in- functions of the A2AR, shifts in inter-related pathways cluding receptor molecules (Cd14, Tlr1, Tlr2, Tlr3 and involved in relaxin signalling, cellular effects of sildenafil Tlr4) and LPS-binding protein (Lbp), transduction mol- and NO signalling (Table 3) support modulation of ecules (Cr3/Mac1 and Cr4), MyD88, TIRAP and cGMP/NOS dependent control, while cardiac arteriopathy MyD88 targets (Il6 and Il1b), MyD88-independent sig- was identified as a pathologic process sensitive to A2AR nal components (Tbk1, Cxcl10, Ccl2 and Ccl5)and KO (Table 4). These rather limited transcriptomic changes downstream JAK-STAT signalling (Fig. S9). in healthy myocardium are consistent with observations in Upregulated TLR and MyD88-dependent and indepen- other tissues. For example, Yu et al. [62] found A2ARKO dent signalling are consistent with changes in rat sepsis alters a very small sub-set of transcripts in healthy stria- [49], though Tlr4, Cd14 and Tirap induction here was tum (implicating A2AR sensitive EGR-2 control), with not apparent in the rat model. Sweeney et al. [63]re- expression changes also modest (a majority ≤twofold). cently reported a novel MyD88-independent path linking

Others report no impact of A2AR KO on myocardial ex- TLR2/TLR4 signalling to Ppargc1a, encoding the mito- pression of RAC1, ERK1/2, p38-MAPK or JNK [14], chondrial biogenesis co-regulator PGC-1α. However, though phospho-activation of the latter kinases was im- despite induction of elements of this path here (includ- paired, potentially reflecting shifts in cAMP/PKA and ing Tlr2, Tlr3, Tlr4 and Irf7), cardiac Ppargc1a was MEKK2 signalling. repressed by LPS, a response potentially exaggerating

mitochondrial dysfunction and countered by A2AR Transcriptomic profile of endotoxemic myocardium activity. Interferon signalling transducing inflammatory/TLR Myocardial injury and dysfunction are critical determi- responses was also upregulated (Fig. 5), including a nants of circulatory changes and mortality with uncon- majority of gene targets and the path of IRF activation trolled inflammation; however, their mechanistic basis is by PRRs (Figs. S5–S7). This entailed induction of poorly defined. Transcriptomic interrogation can reveal membrane (Tlr2, Tlr3 and Tlr4), cytosolic (Ddx58, elements of these complex responses, though there are Ifih1, Eif2ak2 and Oas1) and extracellular (C1q, C3, few analyses of myocardial [41, 42, 49]or C3a and Ptx3) receptors, and downstream mediators Purinergic Signalling (2017) 13:27–49 37

Table 4 Top 20 biological and toxicological functions modified by A2AAR KO in healthy myocardium

Biological pathway P values Genes

Cellular development 7.34E-04–4.5E- Mrap, Ctsk, Sacs, Prkar2b, Ptger3, Nfatc2, Tslp, Map3k2 02 Cellular growth and proliferation 7.34E-04–4.01E- Mrap, Ppbp, Nfatc2, Tslp 02 Haematological system development and 7.34E-04–4.5E- Hp, Ptger3, Ticam1, Ppbp, Nfatc2, Tslp, Map3k2 function 02 Humoral immune response 7.34E-04–4.5E- Nfatc2, Fmod, Tslp 02 Cell-to-cell signalling and interaction 1.53E-03–4.74E- Ank3, Ptger3, Ticam1, Ppbp, Nfatc2, Iqgap1, Tslp, Map3k2 02 Amino acid metabolism 2.55E-03–5.09E- Slc38a1 03 Carbohydrate metabolism 2.55E-03–4.72E- Gnb1, Pon1, Ppbp, Ppa1 02 Cell death 2.55E-03–4.5E- Ppbp, Nfatc2, Tslp, Map3k2 02 Cell morphology 2.55E-03–3.68E- Ank3, Ptger3, Nfatc2, Cnpy2, Iqgap1, Tslp 02 Cellular assembly and organization 2.55E-03–4.01E- Ank3, Ctsk, Nfatc2, Fmod, Cnpy2, Iqgap1, Acta1 02 Cellular compromise 2.55E-03–4.01E- Ank3, Hp, Ctsk, Ptger3, Ppbp 02 Cellular function and maintenance 2.55E-03–1.52E- Hp, Ppbp, Nfatc2, Tslp, Map3k2 02 Cellular movement 2.55E-03–4.58E- Gnb1, Ctsk, Hp, Ticam1, Ppbp, Nfatc2, Iqgap1, Tslp 02 Connective tissue development and function 2.55E-03–3.02E- Ctsk, Rpl22, Ptger3, Pde3b, Ppbp, Cidec, Nfatc2 02 Connective tissue disorders 2.55E-03–5.09E- Nfatc2 03 Developmental disorder 2.55E-03–2.55E- Ctsk 03 Genetic disorder 2.55E-03–4.01E- Ank3, Ctsk, Ptger3, Cidec, Iqgap1, Tslp, Mrap, Gnb1, Pon1, Hp, Prkar2b, Pde3b, Nfatc2, 02 Dgat1, Acta1 Hair and skin development and function 2.55E-03–5.09E- Ptger3, Dgat1 03 Immune cell trafficking 2.55E-03–3.51E- Hp, Ticam1, Ppbp, Nfatc2, Tslp 02 Inflammatory response 2.55E-03–4.67E- Hp, Ptger3, Ticam1, Ppbp, Nfatc2, Fmod, Tslp 02 Molecular toxicological function P values Genes Liver fibrosis 3.01E-03 Hp, Tslp Liver damage 1.43E-02 Ticam1, Tslp Liver steatosis 2.45E-02 Pde3b Cardiac arteriopathy 3.41E-02 Ank3, Cux2, Dgat1, Gk5, Nsmce1, Pde3b, Pon1 Cardiac infarction 3.76E-02 Acta1, Pon1 Liver hepatitis 4.41E-02 Pde3b Hepatocellular carcinoma 4.43E-02 Hp, Iqgap1 Liver steatohepatitis 5.67E-02 Pde3b Renal proliferation 7.90E-02 Tslp Liver inflammation 9.36E-02 Hp Pulmonary hypertension 1.08E-01 Ptger3 Nephrosis 1.10E-01 Pde3b Cardiac congestive cardiac failure 1.63E-01 Pde3b Heart failure 1.63E-01 Pde3b Renal nephritis 2.50E-01 Pde3b

Enriched biological/toxicological functions of A2AR KO sensitive transcripts are listed according to P values or ranges determined by a Fisher’s Exact Test

(Il6, Irf7 and Rantes). These shifts in TLR/CD14, signalling are relevant to temporal expansion of molec- MyD88-dependent and -independent and interferon/IRF ular changes [41, 42] and injury progression in 38 Purinergic Signalling (2017) 13:27–49

Fig. 3 The 25 most induced and 25 most repressed transcripts in hearts Fig. 4 Effects of A2AR KO on the most LPS-responsive cytokines in from young (2–3 month) mice challenged with LPS for 24 h. Mice were wild-type hearts. Mice were injected with 20 mg/kg LPS or saline vehicle, injected with 20 mg/kg LPS or saline vehicle and left ventricular tissue and left ventricular tissue isolated for analysis 24 h later. Data from both isolated for analysis 24 h later. Data from both wild-type and A2ARKO wild-type and A2AR KO hearts are shown for comparison (n =6–8/ hearts are shown for comparison (n =6–8/group). Data are means ± group). Data are means ± S.E.M S.E.M

component of cardio-depression, consistent with reduc- endotoxemia: CD14 [64] and TLR4 [65, 66]bothme- tions observed in β-adrenoceptor expression [73], tissue diate cardiac dysfunction and injury and MyD88 signal- noradrenaline and adrenaline [73] and adrenoceptor ling promotes cardiac hypertrophy [67], inflammation stimulation of cAMP and Ca2+ fluxes [73]. In terms of and injury [68, 69]. These profound responses diverge approaches to inotropic support, coincident depression from in vitro data suggesting dampened/transient im- of adrenoceptor, PKA and Ca2+ signalling may limit pacts of LPS on isolated myocyte NFκBandIκBkinase the value of interventions targeting these effectors. [43, 63]. Abnormalities within the contractile apparatus itself (repressed α-tropomyosin, troponin-T, α-actin and titin Endotoxemic cardio-depression Cardiomyocyte and myo- transcription) may also be relevant. Targeting mitochon- cardial function is LPS-sensitive and depressed in drial dysfunction may more broadly improve cardiac endotoxemia [21, 23, 70, 71](Table1). This has been linked outcomes: repression of Complex I components to abnormalities in myofibrillar Ca2+ sensitivity [70, 71], ad- (Fig. S21) is consistent with Complex I specific dys- renergic control [71–74] and mitochondrial function [75], to- function and ROS generation in mitochondria from gether with cell death [23, 76, 77]. Transient changes in pre- endotoxemic hearts [75, 79]. load may also mediate early reversible depression in vivo [78]. Additional cardio-depressant changes include altered Transcriptomic data reveal a cardio-depressant profile TLR4 and fibroblast factor signalling, suppression of entailing suppression of key determinants of contraction, in- substrate metabolism and IGF-1 signalling (Fig. S22) cluding Ca2+, β-adrenergic and PKA signalling, mitochondri- and induction of inhibitory S100a8/S100a9, Icam1, al function and electromechanical coupling (Figs. S18–S21). Vcam1, Cybb (NOX2), Ptgs2 (COX2) and the TNF-α

Importantly, and despite other impacts, A2ARexpressiondid receptors Tnfr1 and Fas. Marked induction of S100 A8 not influence this cardio-depressant profile nor modify con- and S100 A9 (TLR4 activators promoting endotoxemic tractile depression (Table 1)[23]. injury) has been observed in LPS-treated myocytes, in- Suppression of β-adrenergic and Ca2+ signalling is volving MyD88 and NFκB signalling [80]. Intriguingly, consistent with desensitization in hearts and myocytes two adipokines with opposing actions, relevant to cardi- [71–73], and transcriptional changes in a rat model of ac dysfunction, were among the most responsive to faecal peritonitis [49]. Despite overlap with the latter LPS. Pro-inflammatory and injurious lipocalin-2 (Lcn2) response, the β1- rather than β2-adrenoceptor is de- was the most induced (~600-fold), consistent with pressed in endotoxemic mouse heart (Fig. S18), while changes in rodent and human myocarditis [59], while reductions in AKAP, PP1, PP2A, NCX and L-type Ca2+ anti-inflammatory and cardioprotective adiponectin channel components observed here were not evident in (Adipoq) was the second most repressed. Lipocalin-2 is the peritonitis model. Nonetheless, data collectively im- induced via IκBζ (+13 with LPS) and with heart failure, plicate β-adrenergic dysfunction as a common ischemia and inflammation [81, 82]. Profound induction Purinergic Signalling (2017) 13:27–49 39

Table 5 Top 20 canonical pathways modified by LPS (≥2-fold change, <1 % FDR) in WT hearts

Canonical pathways P Ratio Genes value

Acute phase response signalling 9.12E- 2.02E- Socs3, Rac2, Hamp, Serping1, Nfkbie, Socs2, Cp, Saa2, Serpina3, Jak2, Il6, Rbp1, 09 01 Nr3C1, C1r, Hmox1, Shc1, Nfkbia, Cfb, Lbp, Serpine1, Nfkbib, Saa1, C3, Tnfrsf1a, Myd88, C1s, Rac1, Serpinf1, Cebpb, Stat3, Nfkb2, Hp, Rras2, Il1Rn, C4a, C2 Interferon signalling 1.82E- 4.33E- Ifit3, Oas1, Ptpn2, Mx1, Ifi35, Irf9, Psmb8, Jak2, Tap1, Irf1, Ifitm1, Stat2, Stat1 08 01 Activation of IRF by cytosolic pattern 1.38E- 2.33E- Dhx58, Nfkbie, Zbp1, Irf9, Tbk1, Il6, Nfkb2, Adar, Isg15, Ifih1, Irf7, Nfkbia, Ddx58, recognition receptors 07 01 Stat2, Nfkbib, Stat1, Ifit2 Hepatic fibrosis/hepatic stellate cell 7.94E- 2.01E- Icam1, Lepr, Myh7b (Includes Eg:668,940), Myh11, Ccl5, Il6, Fas, Pgf, Vegfa, Il1r2, activation 07 01 Cxcl3, Igf1, Cyp2e1, Timp1, Lbp, Stat1, Timp2, Egfr, Il4r, Vcam1, Tnfrsf1a, Vegfc, Igfbp5,Nfkb2,Myl7,Csf1,Cd14 Dendritic cell maturation 9.12E- 1.51E- B2m, Rac2, Icam1, Lepr, Nfkbie, Pik3r5, Cd83, Il6, Jak2, Fcgr2b, Fcgr1a, Nfkbia, Hla- 07 01 b, Tlr3, Stat1, Nfkbib, Fcgr3a, Hla-c, Myd88, Tnfrsf1a, Relb, Rac1, Nfkb2, Tlr2, Il1rn, Fcer1g, Stat2, Irf8 Role of macrophages, fibroblasts and 2.57E- 1.34E- Socs3, Rac2, Icam1, Mmp3, Nfkbie, Pik3r5, Jak2, Ccl5, Il6, Il17ra, Fcgr1a, Pgf, Myc, endothelial cells in rheumatoid arthritis 06 01 C1r, Il1r2, Vegfa, Nfkbia, Traf3Ip2, Wif1, Cfb, Plcb1, Tlr3, Nfkbib, Prkd1, Fcgr3a, Calm1, Adamts4, Sele, Vcam1, Myd88, Tnfrsf1a, C1s, Daam1, Rac1, Vegfc, Stat3, Cebpb, Irak3, Tlr2, Hp, Rras2, Il1Rn, Csf1, Cxcl12, Cebpd, Sost, Wnt5a, Irak2 Retinoic acid mediated apoptosis signalling 5.37E- 1.94E- Parp10, Art1, Tnfsf10, Parp3, Parp12, Parp9, Tiparp, Irf1, Parp4, Bid, Parp11, Cflar, 06 01 Parp14 Antigen presentation pathway 6.61E- 2.56E- B2m, Psmb9, Hla-e, Hla-b, Psmb8, Hla-g, Tap1, Tap2, Tapbp, Hla-c 06 01 Role of pattern recognition receptors in 8.32E- 2.12E- Ptx3, Oas1, C3, Oas2, Myd88, Pik3R5, Il6, Nfkb2, Ccl5, Tlr2, Ifih1, Clec7a, Irf7, Syk, recognition of bacteria and viruses 06 01 Ddx58, Eif2ak2, Tlr3 Death receptor signalling 1.29E- 2.34E- Hspb3, Tnfrsf1a, Nfkbie, Tnfsf10, Tbk1, Nfkb2, Fas, Daxx, Nfkbia, Bid, Cflar, Nfkbib, 05 01 Birc3, Birc2, Hspb1 IL-10 signalling 3.24E- 2.14E- Ccr1, Socs3, Il4r, Nfkbie, Il6, Stat3, Nfkb2, Fcgr2b, Il1r2, Hmox1, Nfkbia, Il1rn, Cd14, 05 01 Lbp, Nfkbib Role of RIG1-like receptors in antiviral 6.61E- 1.96E- Dhx58, Ifih1, Irf7, Nfkbia, Nfkbie, Ddx58, Tbk1, Nfkb2, Nfkbib, Trim25 innate immunity 05 01 IL-6 signalling 1.00E- 1.94E- Hspb3, Tnfrsf1a, Nfkbie, Il6, Nfkb2, Stat3, Cebpb, Jak2, Il1r2, Shc1, Nfkbia, Rras2, 04 01 Il1rn, Cd14, Lbp, Nfkbib, Tnfaip6, Hspb1 IL-8 signalling 1.02E- 1.45E- Rac2, Icam1, Cxcl1, Pik3r5, Pgf, Eif4ebp1, Vegfa, Hmox1, Gng11, Gna13, Nfkbib, 04 01 Prkd1, Egfr, Vcam1, Rhoc, Rac1, Vegfc, Irak3, Cstb, Myl7, Myl9 (Includes Eg:98,932), Bcl2l1, Itgb2, Rras2, Ccnd2, Itgam, Irak2 Bladder cancer signalling 1.26E- 1.89E- Fgf16, Mmp3, Fgf9, Mmp14, Mmp15, Vegfc, Pgf, Myc, Vegfa, Rras2, Mmp8, Fgf12, 04 01 Cdkn1a, Chd1 (Includes Eg:1105), Rps6Ka5, Fgf7, Egfr Pathogenesis of multiple sclerosis 1.45E- 5.56E- Cxcl10, Ccr1, Cxcl9, Ccl5, Cxcl11 04 01 Role of PKR in interferon induction and 1.58E- 2.39E- Nfkbia, Tnfrsf1a, Nfkbie, Bid, Nfkb2, Eif2ak2, Tlr3, Stat1, Nfkbib, Fcgr1a, Irf1 antiviral response 04 01 JAK/Stat signalling 2.00E- 2.19E- Rac2, Socs3, Pias2, Socs2, Rac1, Pik3r5, Stat3, Jak2, Shc1, Rras2, Cdkn1a, Cish, Stat2, 04 01 Stat1 Complement system 2.09E- 2.5E- C1R, Cfd, Serping1, C3, C1S, Cfb, C4a, Masp1, C2 04 01 Glucocorticoid receptor signalling 2.14E- 1.25E- Rac2, Icam1, Nfkbie, Pik3R5, Pbx1, Ccl5, Jak2, Il6, Fcgr1a, Nr3c1, Il1R2, Shc1, Cxcl3, 04 01 Hspa4, Nfkbia, Ar, Ccl13, Cdkn1c, Serpine1, Stat1, Fkbp5, Nfkbib, Sra1, Vcam1, Ccnh, Sele, Rac1, Stat3, Cebpb, Bcl2l1, Rras2, Il1rn, Cdkn1a, Fkbp4, Nr3c2

may promote dysfunction given its inflammatory [83], [85, 86], thus downregulation is also likely to promote pro-death [82, 84] and mitochondrial actions [84]. inflammation and sensitize myocardium to oxidative-

Current data suggest the A2AR may beneficially limit stress and cell death. Curiously, Adipoq repression was LPS-dependent IκBζ induction to suppress injurious reduced with A2AR KO, suggesting a positive influence lipocalin-2. Highly repressed adiponectin exerts anti-in- of A2AR activity on this response. These and other flammatory, anti-oxidant and cardioprotective effects changes relevant to pathogenesis of cardiac depression 40 Purinergic Signalling (2017) 13:27–49 Purinergic Signalling (2017) 13:27–49 41

Fig. 6 Schematic of TLR, IL-1, IL-6 and interferon receptor activation of NFκBandJAK- STAT signalling and transcriptional control. The effects of A2AR KO- on LPS- dependent gene changes are highlighted. These pathways are critical to cellular responses to LPS and orchestration of inflammation. Specific effects of A2AR activity (based on effects of receptor KO) on transcriptional responses to LPS are highlighted (red—increased expression; blue—reduced expression)

are discussed further in the online Supplementary cardiac IL-6 signalling. Functional annotation of A2AR material. sensitive transcripts does support modulation of acute phase response, JAK-STAT, MAPK, PRR and NFκB

A2AR modulation of the endotoxemic transcriptome pathways (Tables 7, S6 and S10). Figure 6 summarizes these key changes, highlighting impacts of A2ARKO. Despite well-established anti-inflammatory effects of Through modifying a small number of key transcript

acute A2AR agonism, the myocardial transcriptomic re- responses (Nfkbie, Nfkbiz, Il6, Lcn2, Stat1, Cdkn1a and sponse to endotoxemia was largely insensitive to A2AR Rras2), A2AR activity may limit expansion of injurious KO (Figs. 3 and 4;Tables7 and S9). Rather than a inflammation in endotoxemic myocardium. In contrast,

broadly suppressive impact of A2AR activity, only the cardio-depressant molecular profile appears insensi- 10 % of the transcriptomic response was modified by tive to A2AR KO. KO. This selective outcome is consistent with data for — cytokine/chemokine transcription and release the ma- NFκBsignallingThe A2ARrepressesNFκBsignallingvia jority of these responses were also unaltered by A2AR multiple mechanisms in non-cardiac cells, including phos- deletion, which specifically modified Il6, Cxcl2, Cxcl5 phorylation-/SUMOylation-dependent IκB degradation and and Csf3 induction (Fig. 4) and circulating IL-5, hapto- modulation of the SCF-E3 ubiquitin ligase complex [58].

globin and CRP levels (Figs. 1 and 2). These responses The absence of A2ARs did modify the NFκBpathin are nonetheless relevant to myocardial outcome: IL-6 endotoxemic myocardium (Fig. 6), amplifying pro- initiates and expands inflammatory signalling, and inflammatory Nfkbiz (IκBζ) induction while countering inhib- CXCL2 and CXCL5 both participate in cardiac inflam- itory Nfkbie (IκBε) induction. Enhanced transcription of IκBζ

mation and remodelling. Important IL-6 target genes and its targets Lcn2 and Il6 with A2AR KO suggests the re- were highly induced with LPS, including Bcl2l1, ceptor may suppress inflammation by inhibiting IκBζ induc- Cebpb, Irf1, JunB, Lbp, Socs3 and Timp1. By suppress- tion and its transcriptional actions, while promoting IκBε in- ing IL-6 transcription, potentially through IκBζ induc- duction to limit nuclear translocation of NFκB(Fig.6).

tionandSTAT1repression,A2AR activity may limit In contrast, A2AR KO also reduced induction of molecules promoting NFκB signalling (Fig. 6): Eif2ak2/Pkr inhibits IκBα expression and enhances NFκB signaling Fig. 5 Impact of 24 h LPS challenge on cardiac transcription of the TLR and IFN-β expression; Btrc promotes degradation of (upper panel) and interferon signalling paths (lower panel) in wild-type IκBα and Rac2 promotes cytokine-triggered NFκB mice (n =6–8/group). Induced transcripts highlighted in red and γ repressed in green. Shaded transcripts are altered by LPS by less than signalling and IFN- expression. It is unclear what the the threshold value balance of these changes might be, though repression 42 Purinergic Signalling (2017) 13:27–49

Table 6 Top canonical responses to LPS modified by A2AAR KO (the 15 most promoted or inhibited by A2AAR KO are shown)

Canonical Pathways P value Ratio Genes

LPS responses enhanced by A2AR KO (countered by A2ARactivity) Acute phase response signalling 7.41E- 5.06E- Hpx, Hp, Rras2, Pik3r1, Cp, Il6, Serpine1, Nr3c1, Orm1 04 02 Glucocorticoid receptor signalling 9.33E- 3.90E- Hspa4, Cxcl3, Rras2, Pik3r1, Cdkn1a, Tgfb2, Il6, Stat1, Serpine1, Hspa2, 04 02 Nr3c1 Glioma invasiveness signalling 1.29E- 8.47E- Timp4, Rras2, Pik3r1, Rhoj, Itgb3 03 02 Germ cell-sertoli cell junction signalling 1.45E- 5.00E- Rras2, Tuba8, Pak6, Map3k6, Pak3, Pik3r1, Tgfb2, Rhoj 03 02 Angiopoietin signalling 3.16E- 6.58E- Grb14, Rras2, Pak6, Pak3, Pik3r1 03 02 ILK signalling 3.55E- 4.26E- Ppp2r3a, Pik3r1, Vegfc, Atf4, Rhoj, Itgb6, Pgf, Itgb3 03 02 Ephrin receptor signalling 3.98E- 4.04E- Rras2, Pak6, Pak3, Vegfc, Atf4, Epha4, Efnb3, Pgf 03 02 ERK/MAPK signalling 4.37E- 4.10E- Rras2, Pak6, Pak3, Ppp2r3a, Ppp1R3c, Pik3r1, Atf4, Stat1 03 02 Pancreatic adenocarcinoma signalling 4.47E- 5.08E- Pik3r1, Cdkn1a, Tgfb2, Vegfc, Stat1, Pgf 03 02 HER-2 signalling in breast cancer 5.13E- 6.17E- Rras2, Pik3r1, Cdkn1a, Itgb6, Itgb3 03 02 Macrophage NO & ROS production 7.08E- 3.74E- Map3k6, Ppp2r3a, Ppp1r3c, Pik3r1, Rhoj, Irf8, Stat1 03 02 Hepatic fibrosis/stellate cell activation 1.05E- 4.48E- Cxcl3, Tgfb2, Vegfc, Il6, Stat1, Pgf 02 02 Circadian rhythm signalling 1.12E- 8.57E- Per3, Arntl, Atf4 02 02 HIF1α signalling 1.38E- 4.55E- Rras2, Pik3r1, Vegfc, Slc2a4, Pgf 02 02 JAK/Stat signalling 1.41E- 6.06E- Rras2, Pik3r1, Cdkn1a, Stat1 02 02 LPS responses inhibited by A2AR KO (promoted by A2AR activity) PXR/RXR activation 6.46E- 4.4E-02 Rac2, Prkar2b, Cyp2b6 04 Nicotinate and nicotinamide metabolism 3.98E- 2.94E- Cilp, Art5, Eif2ak2, Bst1 03 02 Amyloid processing 4.57E- 5.08E- Rac2, Prkar2b, Capn10 03 02 Glutamate receptor signalling 5.89E- 4.29E- Glul, Slc38a1, Homer1 03 02 AMPK signalling 9.55E- 2.40E- Rac2, Prkar2b, Adipoq, Eif4ebp1 03 02 PRRs in recognition of bacteria & viruses 9.77E- 3.66E- C3, Ddx58, Eif2ak2 03 02 NF-κB signalling 1.15E- 2.58E- Rac2, Nfkbie, Btrc, Eif2ak2 02 02 NF-κB activation by viruses 1.23E- 3.61E- Rac2, Nfkbie, Eif2ak2 02 02 Inhibition of angiogenesis by TSP1 1.55E- 5.56E- Rac2, Sdc1 02 02 Role of RIG1-like receptors in antiviral innate immunity 1.66E- 3.92E- Nfkbie, Ddx58 02 02 FXR/RXR activation 1.78E- 2.91E- Rac2, Sdc1 02 02 TR/RXR activation 1.78E- 3.03E- Rac2, Ucp1 02 02 PPARα/RXRα activation 1.86E- 2.22E- Prkar2b, Nfkbie, Adipoq 02 02 RANK signalling in osteoclasts 1.95E- 3.06E- Rac2, Nfkbie, Map3k2 02 02 Fcγ receptor-mediated phagocytosis in macrophages and 2.04E- 2.97E- Hmox1, Rac2, Hck monocytes 02 02

Canonical pathways modified by LPS, ranked according to P-values determined by Fisher’s Exact Test. Also shown is the ratio reflecting the number of molecules in a given path that meet cut-off criteria for differential expression, divided by the total number of molecules in the path Purinergic Signalling (2017) 13:27–49 43

Table 7 Functional groupings of genes for which LPS responses Functional grouping P value Number of genes were modified by ≥1.5-fold (induction or repression) by A2AR LPS responses enhanced by A2AR KO (countered by A2AR activity) KO Molecular and cellular functions Cellular Movement 1.10E-08–5.10E-03 58 Cell-To-Cell Signalling and Interaction 1.78E-06–5.40E-03 46 Cell Death 1.13E-05–4.87E-03 74 Cellular Growth and Proliferation 1.30E-05–5.40E-03 58 Cell Cycle 2.22E-05–4.86E-03 30 Physiological system development and function Haematological System Development and Function 3.17E-07–5.40E-03 53 Haematopoiesis 3.17E-07–4.79E-03 36 Tissue Morphology 3.17E-07–4.79E-03 38 Immune Cell Trafficking 3.90E-07–5.10E-03 37 Cardiovascular System Development and Function 1.54E-06–4.25E-03 24 Disease and disorders Cancer 3.71E-08–5.03E-03 82 Immunological Disease 9.17E-08–3.81E-03 72 Skeletal and Muscular Disorders 7.90E-07–3.81E-03 77 Inflammatory Response 1.78E-06–5.05E-03 46 Haematological Disease 2.44E-06–3.68E-03 37

LPS responses inhibited by A2AR KO (promoted by A2AR activity) Molecular and cellular functions Carbohydrate Metabolism 2.24E-05–2.33E-02 6 Molecular Transport 2.38E-05–2.61E-02 24 Small Molecule Biochemistry 2.38E-05–2.87E-02 34 Lipid Metabolism 4.28E-05–2.87E-02 14 Cellular Compromise 6.16E-05–2.61E-02 11 Physiological system development and function Cardiovascular System Development and Function 4.28E-05–2.97E-02 5 Organ Development 4.28E-05–2.33E-02 7 Organismal Functions 9.49E-05–2.61E-02 4 Haematological System Development and Function 2.54E-04–3.11E-02 13 Lymphoid Tissue Structure and Development 2.54E-04–2.61E-02 6 Disease and disorders Inflammatory Response 6.16E-05–3.11E-02 15 Nutritional Disease 8.22E-05–6.58E-03 10 Genetic Disorder 1.34E-04–3.00E-02 30 Developmental Disorder 2.54E-04–2.61E-02 14 Neurological Disease 2.54E-04–2.61E-02 30

Functional groupings of LPS-responsive cardiac transcripts modified by A2AR KO (enhanced or repressed) by a factor of ≥1.5-fold. Functional groups derived from IPA analysis are categorized into molecular and cellular functions, physiological system development and function, and disease and disorders, and ranked according to P value ranges determined by a Fisher’s Exact Test. Total numbers of involved genes are also shown of the distal transcriptional effector IκBζ is predicted to receptor activity might selectively modify injurious rather limit effects of up-stream changes. Failure of A2ARKOto than protective aspects of NFκB signalling. modify changes in genes recently implicated in cardioprotective effects of NFκB signalling [47](includ- JAK-STAT signalling In non-cardiac cells, A2AR agonism ing induction of Ptx3, Plscr1, Sfi1 and Igfbp3 and repres- also inhibits JAK-STAT signalling, via control of SOCS tran- sion of Car3, Dkk3, ai605517 and Grhl2) suggests scription [58] and ubiquitination/degradation of JAK- 44 Purinergic Signalling (2017) 13:27–49

response yet not associated TLR and interferon signalling.

Novel effects of A2ARKOThe impact of A2ARactivityis limited primarily to canonical elements of endotoxemia (Tables 6, S10 and S11); however, several of the more

A2AR-sensitive transcriptional responses to LPS hint at novel effects of A2AR signalling, including modulation of insulin- sensitivity, hypertrophic growth/remodelling and cardiac rhythmicity, together with influencing angiogenesis and vascular control. Fig. 7 Comparison of expression values for select transcripts assessed Disruption of insulin-dependent glucose metabolism is an via array analysis and quantitative real-time PCR. Expression ratios are important consequence of endotoxemia, and three of the most relative to values in wild-type (WT) hearts treated with normal saline (NS) and are thus shown for WTs treated with LPS for 24 h (WT LPS) A2AR-sensitive transcripts (Ptprf, Glut4/Slc2a4 and Glut12/ and A2AR KO hearts untreated (KO NS) or treated with LPS (KO LPS). Slc2a12) govern insulin-dependent glucose uptake Data are means ± S.E.M, n =6–8/group (Table S9). Endotoxemic suppression of Glut4 and the sec- ondary insulin-sensitive transporter Glut12 and the induction of Ptprf (encoding protein tyrosine phosphatase, receptor type phosphorylated STATs [87]. Despite no evidence for A2AR F; implicated in insulin-resistance) were exaggerated >two- modulation of Socs3 (induced with LPS unaltered by KO), fold with A2ARKO(TableS9). Endotoxemic suppression of A2AR deletion did modify the JAK-STAT response, exagger- Glut4 and glucose uptake is reported in skeletal and cardiac ating induction of both Stat1 and the key activator Il6. Since muscle [89]. Conversely, increased expression of Glut4 im- STAT1 is crucial to LPS-induced apoptosis in non-cardiac proves glucose uptake and limits myocardial dysfunction in cells [88], and mediates injury with ischemic insult [39], endotoxemia [89]. These responses to A2AR KO suggest reg- A2AR-dependent suppression is predicted to be protective. ulatory influences of A2AR activity on glucose handling in Deletion of A2ARs also enhanced endotoxemic induction of endotoxemic tissue. Cdkn1a/p21Cip1/Waf1 (modulator of STAT1-dependent apo- Other highly A2AR-sensitive transcriptional responses γ ptosis and IFN- /STAT signalling, linked to poor sepsis out- suggest A2AR modulation of myocardial remodelling, in- comes [49]) and the signal transducer Rras2. These data col- cluding shifts in Asb14 and Asb15, Ca3 and Ca4 and lectively support beneficial A2ARmodulationofJAK-STAT Dnmt3a and Srpk2. Endotoxemic repression of transcripts activation in endotoxemic myocardium (Fig. 6). for 2 ankyrin repeat and SOCS box (ASB) proteins, Asb14 and Asb15, was substantially exaggerated with

A2AR KO. The ASB family bind and target proteins for Select impacts on acute phase response vs. TLR and inter- degradation and regulate skeletal muscle development, feron signalling The acute phase response is triggered by while roles in heart are unclear. The ASB15 protein en- IL-1, IL-6 and TNF-α and transduced via NFκΒ and hances skeletal muscle protein synthesis [90]anddelays JAK-STAT signals. Additional to effects of KO on Il6 differentiation, potentially via modulating MAPK and and JAK-STAT and NFκBpaths,A2AR activity appears PI3K/Akt signalling [91]. This control may be important to counter induction of key gene targets (Serpine1, Il6, in adaptive response to muscle load/activity. Carbonic Cp, Hpx, Hp and Orm1). Conversely, A2AR activity was anhydrases are also implicated in myocardial hypertrophy, also associated with greater induction of target genes C3 with endotoxemic Car4 induction (the dominant cardiac and Hmox and reduced induction of the glucocorticoid isoform) exaggerated and Car3 downregulation was receptor inhibiting this inflammatory response. While inhibitedinA2AR KO hearts. The myocardial roles of mixed, collective shifts in JAK-STAT/NFκBpathssup- these enzymes are only beginning to be unravelled, port transcriptional suppression of the acute phase re- though they may promote hypertrophic responses by fa- + + sponse via A2AR activity. However, while TLR/ cilitating Na -H and Cl-HCO3 exchanger over-activities, interferon paths were among the most highly responsive while evidence from other cells suggests CAR3 may mod- to LPS, A2AR KO did not substantially modify these ify oxidative stress and apoptosis. Similarly, Srpk2 induc- responses (augmenting induction of MyD88-responsive tion was augmented in the absence of A2ARs, encoding a Irf8 and altering downstream NFκB signalling). Major serine/arginine (SR) protein kinase (SRPK) that phosphor- changes in interferon signalling were also largely un- ylates SR domain-containing proteins within modified by A2ARKO(Fig.6), suggesting select im- interchromatin granule clusters/nuclear speckles to regu- pacts of A2AR activity on elements of the acute phase late pre-mRNA splicing. The SRPK2 protein also plays an Purinergic Signalling (2017) 13:27–49 45 important role in cell proliferation and apoptosis. RT-qPCR confirmation of microarray-detected responses Additionally, downregulation of the methyltransferase transcript Dnmt3a, governing growth and function of em- Data confirm agreement between transcript expression de- bryonic myocytes [92],appearstobecounteredbyA2AR termined via RT-qPCR and microarray methods (Fig. 7), activity, which may also limit cardiac fibrosis/remodelling with PCR further highlighting distinct transcriptional ef-

[93]. Collectively, this suite of A2AR responsive changes fects of A2AR KO: in some cases, KO eliminates re- may limit myocardial hypertrophy and remodelling responses to LPS (Acta1); alters baseline yet not LPS- sponses to inflammatory insult. This is consistent with dependent expression (Amid or Dbp); enhances induction functional groupings sensitive to A2AR KO, including de- (Txnip)orrepression(Cidec) in response to vehicle and terminants of cell cycle, growth, proliferation and death LPS or reduces gene expression in the vehicle group and cardiovascular development (Table 7) and shifts in while negating further changes with LPS (Eif4ebp2 and canonical hypertrophy pathways (Tables S10 and S11). Slc38a1). Confirming the microarray approach, some of

Curiously, A2AR activity also limited LPS induction of these responses are also functionally relevant. For exam- Hcn1 (Table S9). The HCN proteins mediate the pacemaker ple, Txnip encodes a stress-responsive protein inhibiting (funny) current governing cardiac automaticity/excitability. the anti-oxidant/signalling molecule thioredoxin. While HCN4 is the most highly expressed, HCN1 is signifi- Reductions in TXNIP inhibit vascular inflammation and cantly expressed within the conduction network, contributes TNF-α signalling [97], TXNIP dysregulation promotes in- to cardiac rhythmicity [94] and is up-regulated in hypertrophy flammatory disease [98] and elevations in TXNIP facili- and heart failure. Endotoxemic induction may thus promote tate apoptosis [99]. Inhibition of Txnip inductionbyA2AR arrhythmicity, while A2AR activity appears to suppress this activity may thus enhance protective thioredoxin change. functionality.

Finally, A2AR KO modified a broader range of vascular control and growth pathways in endotoxemic (vs. Study limitations healthy) hearts. Vascular dysfunction is a critical to organ damage and mortality and may involve NOS over- Several study limitations are worth noting. While essential activity in some vascular beds. Deletion of the A2AR genesis of cardiac injury is studied and understood within modified NO signalling together with renin-angiotensin the in situ organ, this entails experimental limitations in- signalling (Table S10), while cardiac arteriopathy was herent to in vivo studies of organ dysfunction in sepsis/ identified as a pathological process sensitive to KO endotoxemia [15, 16, 20, 21, 24, 41, 42, 49]. Organ injury (Table S11). These responses support influences of in these settings not only involves intrinsic organ-specific

A2AR signalling on vascular pathology. We have previ- mechanisms but also influenced by extrinsic factors, in- ously shown endotoxemia impairs coronary hyperaemia, cluding shifts in systemic inflammation, haemodynamics a dysfunction mimicked by KO of (and potentially in- and neuroendocrine influences (with relative impacts of volving) the A2AR[23]. Additionally, there is an appar- these factors difficult to delineate). Although we assess ent coronary ‘over-supply’ relative to myocardial de- the intrinsic myocardial dysfunction in ex vivo tissue (to- mand in endotoxemic hearts (Table 1), owing to 25– gether with in vivo markers of cardiac damage), we can-

30 % lower contractile function without reductions in not isolate potential influences of A2ARKOoncardiac coronary perfusion. The absence of coronary coupling loading, neurohumoral factors or indeed systemic inflam- to ventricular activity, described by us in this and other mation. That said, while A2AR KO augmented some in- models [95, 96], suggests additional coronary dysregu- flammatory markers, systemic and cardiac cytokine re- lation (though this effect is neither replicated nor mod- sponses were largely insensitive to KO and the myocardi- ified by A2AR KO). Multiple paths/mediators regulating al response was very selectively modified, indicating dis- angiogenesis were also sensitive to A2ARKOin tinct effects of A2AR activity on cardiac injury processes. endotoxemic heart, including shifts in VEGF, HIF-1α, We are also unable to detail cell-specific origins of myo- angiopoietin, TGF-β, GM-CSF, PDGF and IGF-1 path- cardial transcriptional changes. Migrating inflammatory ways (Table S10) and key regulatory molecules (includ- cells could influence the transcriptomic profile for intact ing exaggerated induction of Il6, Pgf, Tgfb2, Tgfb2r, myocardium, though, as detailed in the Supplementary Angptl4 and Amotl1; exaggerated suppression of Vegfc material, the expression profile of endotoxemic myocardi- and reduced suppression of Qk)(TableS9). While the al tissue is inconsistent with major contamination from

A2B receptor is more broadly implicated in control of such cells. An added limitation relates to the intriguing angiogenesis, these changes suggest A2AR-dependent observation of both age- and sex-dependent mortality ef- processes may influence angiogenesis in the context of fects of LPS and A2ARs (Fig. S3). This unfortunately uncontrolled inflammation. precluded any detailed interrogation of these outcomes 46 Purinergic Signalling (2017) 13:27–49 due to poor survival in older KO mice. Future work might References focus specifically on responses to LPS/sepsis in clinically relevant aged cohorts, correlating age-dependent transcriptomic and phenotypic outcomes. Finally, while 1. Merx MW, Weber C (2007) Sepsis and the heart. Circulation 116: 793–802 profound transcriptional changes observed in the current 2. Balija TM, Lowry SF (2011) Lipopolysaccharide and sepsis- profiling study are predicted to translate to functionally associated myocardial dysfunction. 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Ribé D, Sawbridge D, Thakur S, Hussey M, Ledent C, Kitchen I Lung and Blood Institute grants HL-74001 (RRM), HL-027339 (SJM) et al (2008) Adenosine A2A receptor signaling regulation of car- and HL-48839 (PAH), an American Heart Association Southeast Affiliate diac NADPH oxidase activity. Free Radic Biol Med 44:1433– Grant-in-Aid (PAH), a grant from the National Health and Medical 1442 Research Council of Australia (JPH) and the American Lebanese 14. Xi J, McIntosh R, Shen X, Lee S, Chanoit G, Criswell H et al Syrian Associated Charities (RRM). We gratefully acknowledge the tech- (2009) Adenosine A2A and A2B receptors work in concert to induce nical assistance of Kiel J. Headrick and Mary A. Cerniway. a strong protection against reperfusion injury in rat hearts. J Mol Cell Cardiol 47:684–690 Authors’ contributions Conceived and designed the experiments: 15. He X, JL H, Li J, Zhao L, Zhang Y,Zeng YJ et al (2013) A feedback MER, KJA, JPH, RRM, SJM. Performed the experiments: MER, KJA, loop in PPARγ-adenosine A2A receptor signaling inhibits inflam- RRM, BT. Analysed data: KJA, MER, JPH, RRM, BT, LD. Generated mation and attenuates lung damages in a mouse model of LPS- and characterized KO mice: CL, PAH. Contributed reagents/materials/ induced acute lung injury. Cell Signal 25:1913–1923 analytical tools: RRM, CL, PAH, KJA, JPH. Wrote the paper: KJA, MER, JPH, RRM, LMD, SJM. 16. Gonzales JN, Gorshkov B, Varn MN, Zemskova MA, Zemskov EA, Sridhar S et al (2014) Protective effect of adenosine receptors against lipopolysaccharide-induced acute lung injury. Am J Physiol Compliance with ethical standards Lung Cell Mol Physiol 306:L497–L507 17. Odashima M, Otaka M, Jin M, Komatsu K, Wada I, Matsuhashi T Conflict of interest The authors declare that they have no conflict of et al (2005) Selective A2A adenosine agonist ATL-146e attenuates interest. acute lethal liver injury in mice. 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